The 5-fluoropyrimidines comprise an important class of antitumor compounds. One derivative of this class, 5-fluoro,2'-deoxycytidine (FdC) is known to have potent.sup.1-5 1/ and selective.sup.6-8 cytostatic properties. It has been established that FdC can be kinased and deaminated to FdUMP via two distinct metabolic pathways resulting in the inhibition of thymidylate synthase.sup.9. Due to the increased activity of both cytidine and deoxycytidine deaminases present in many human tumor cells, it has been proposed that FdC might have superior properties as an antitumor agent in man.sup.8. FNT .sup.1/ See the Bibliography preceding the claims.
FdC was first synthesized by Fox et al., via aminolysis of 4-thio,5-fluoro,2'-deoxyuridine.sup.14. FdC has also been prepared by coupling the fluorinated pyrimidine with a suitably derivatized deoxyribose.sup.15. Both of these procedures have the disadvantages of modest yield and complexity of products. Robins et al. have presented a method for direct fluorination of deoxycytidine.sup.16. While FdC was provided in high yield with few side products, the required fluorination reagents are potentially toxic and explosive.
Accordingly, one aspect of the invention comprises a novel method to provide FdC in high yield under mild conditions.
It is also known that FdC has important biochemical properties unrelated to the inhibition of thymidylate synthetase. FdC can induce cellular differentation.sup.10 and cause dramatic decreases in DNA 5-methylcytosine levels.sup.10,11. FdC is incorporated into cellular DNA and it has been shown that the level of FdC incorporation is proportional to cytotoxicity.sup.11. Recently, interest in FdC has been renewed as it has been shown that oligonucleotides containing FdC are potent mechanism based inhibitors of both procaryotic.sup.12 and eucaryotic.sup.13 DNA (cytosine-5) methyltransferases.
Physical and biochemical studies aimed at elucidation of the mechanisms which explain the biological properties of FdC require methods for synthesis of FdC in oligonucleotides at unique sites. Previously, FdC has been introduced into oligonucleotides by incorporation of FdCTP by DNA polymerase.sup.4,5. This method suffers from the disadvantage that FdC would be incorporated at multiple sites and quantities sufficient for physical studies are not practically attainable. It has been reported that attempts to prepare FdC containing oligonucleotides via standard methods for deoxycytidine incorporation fail because the N-benzylated derivative of FdC decomposes under normal synthetic conditions.sup.4.
Accordingly, another aspect of this invention provides a practical method for preparing oligonucleotides containing FdC.